A droplet actuator typically includes one or more substrates configured to form a surface or gap for conducting droplet operations. The one or more substrates establish a droplet operations surface or gap for conducting droplet operations and may also include electrodes arranged to conduct the droplet operations. The droplet operations substrate or the gap between the substrates may be coated or filled with a filler fluid that is immiscible with the liquid that forms the droplets.
Droplet actuators are used to conduct a variety of molecular protocols, such as amplification of nucleic acids and nucleic acid sequencing. Nucleic acid amplification and sequencing methods are used in a wide variety of settings, such as medical diagnostics and detection of sequence variations. Pyrosequencing is a sequencing-by-synthesis method in which a primed DNA template strand is sequentially exposed to one of four nucleotides in the presence of DNA polymerase. The nucleotides are sequentially added one by one according to a fixed order dependent on the template and pre-determined by the user. Consequently, the operator must enter the order of the nucleotides before the instrument analysis begins. Thus, the operator needs to know the sequence of the sample in order to be able to enter the dispensation order in advance. In certain instances, an optimal dispensation order may be difficult to determine or is not known. As a consequence, many unnecessary dispensations may be made where no nucleotides are incorporated. Unnecessary dispensations are a waste of time and resources and contribute to a decline in accuracy as the sequencing reaction progresses. Recent advances in pyrosequencing technology include methods for using sequence information in a database of alleles and adapting the dispensation order in real-time during the analysis. However, this approach is limited to statistical selection of the next nucleotide to add based only on nucleotide frequency in a database. Therefore, there is a need for improved pyrosequencing methods for rapid, accurate and efficient sequencing of nucleic acids.